TNF Ligand Family
The cytokine known as tumor necrosis factor-α (TNFα; also termed cachectin) is a protein secreted primarily by monocytes and macrophages in response to endotoxin or other stimuli as a soluble homotrimer of 17 kD protein subunits (Smith, R. A. et al., J. Biol. Chem. 262:6951–6954 (1987)). A membrane-bound 26 kD precursor form of TNF has also been described (Kriegler, M. et al., Cell 53:45–53 (1988)).
Accumulating evidence indicates that TNF is a regulatory cytokine with pleiotropic biological activities. These activities include: inhibition of lipoprotein lipase synthesis (“cachectin” activity) (Beutler, B. et al., Nature 316:552 (1985)), activation of polymorphonuclear leukocytes (Klebanoff, S. J. et al, J. Immunol. 136:4220 (1986); Perussia, B., et al., J. Immunol. 138:765 (1987)), inhibition of cell growth or stimulation of cell growth (Vilcek, J. et al., J. Exp. Med. 163:632 (1986); Sugarman, B. J. et al., Science 230:943 (1985); Lachman, L. B. et al., J. Immunol. 138:2913 (1987)), cytotoxic action on certain transformed cell types (Lachman, L. B. et al., supra; Darzynkiewicz, Z. et al., Canc. Res. 44:83 (1984)), antiviral activity (Kohase, M. et al., Cell 45:659 (1986); Wong, G. H. W. et al., Nature 323:819 (1986)), stimulation of bone resorption (Bertolini, D. R. et al., Nature 319:516 (1986); Saklatvala, J., Nature 322:547 (1986)), stimulation of collagenase and prostaglandin E2 production (Dayer, J.-M. et al., J. Exp. Med. 162:2163 (1985)); and immunoregulatory actions, including activation of T cells (Yokota, S. et al., J. Immunol. 140:531 (1988)), B cells (Kehrl, J. H. et al., J. Exp. Med. 166:786 (1987)), monocytes (Philip, R. et al., Nature 323:86 (1986)), thymocytes (Ranges, G. E. et al., J. Exp. Med. 167:1472 (1988)), and stimulation of the cell-surface expression of major histocompatibility complex (MHC) class I and class II molecules (Collins, T. et al., Proc. Natl. Acad. Sci. USA 83:446 (1986); Pujol-Borrel, R. et al., Nature 326:304 (1987)).
TNF is noted for its pro-inflammatory actions which result in tissue injury, such as induction of procoagulant activity on vascular endothelial cells (Pober, J. S. et al., J. Immunol. 136:1680 (1986)), increased adherence of neutrophils and lymphocytes (Pober, J. S. et al., J. Immunol. 138:3319 (1987)), and stimulation of the release of platelet activating factor from macrophages, neutrophils and vascular endothelial cells (Camussi, G. et al., J. Exp. Med. 166:1390 (1987)).
Recent evidence implicates TNF in the pathogenesis of many infections (Cerami, A. et al., Immunol. Today 9:28 (1988)), immune disorders, neoplastic pathology, e.g., in cachexia accompanying some malignancies (Oliff, A. et al., Cell 50:555 (1987)), and in autoimmune pathologies and graft-versus host pathology (Piguet, P.-F. et al., J. Exp. Med. 166:1280 (1987)). The association of TNF with cancer and infectious pathologies is often related to the host's catabolic state. A major problem in cancer patients is weight loss, usually associated with anorexia. The extensive wasting which results is known as “cachexia” (Kern, K. A. et al. J. Parent. Enter. Nutr. 12:286–298 (1988)). Cachexia includes progressive weight loss, anorexia, and persistent erosion of body mass in response to a malignant growth. The cachectic state is thus associated with significant morbidity and is responsible for the majority of cancer mortality. A number of studies have suggested that TNF is an important mediator of the cachexia in cancer, infectious pathology, and in other catabolic states.
TNF is thought to play a central role in the pathophysiological consequences of Gram-negative sepsis and endotoxic shock (Michie, H. R. et al., Br. J. Surg. 76:670–671 (1989); Debets, J. M. H. et al., Second Vienna Shock Forum, p.463–466 (1989); Simpson, S. Q. et al., Crit. Care Clin. 5:27–47 (1989)), including fever, malaise, anorexia, and cachexia. Endotoxin is a potent monocyte/macrophage activator which stimulates production and secretion of TNF (Kornbluth, S. K. et al., J. Immunol. 137:2585–2591 (1986)) and other cytokines. Because TNF could mimic many biological effects of endotoxin, it was concluded to be a central mediator responsible for the clinical manifestations of endotoxin-related illness. TNF and other monocyte-derived cytokines mediate the metabolic and neurohormonal responses to endotoxin (Michie, H. R. et al., N. Eng. J. Med. 318:1481–1486 (1988)). Endotoxin administration to human volunteers produces acute illness with flu-like symptoms including fever, tachycardia, increased metabolic rate and stress hormone release (Revhaug, A. et al., Arch. Surg. 123:162–170 (1988)). Elevated levels of circulating TNF have also been found in patients suffering from Gram-negative sepsis (Waage, A. et al., Lancet 1:355–357 (1987); Hammerle, A. F. et al., Second Vienna Shock Forum p. 715–718 (1989); Debets, J. M. H. et al., Crit. Care Med. 17:489–497 (1989); Calandra, T. et al., J. Infec. Dis. 161:982–987 (1990)).
Passive immunotherapy directed at neutralizing TNF may have a beneficial effect in Gram-negative sepsis and endotoxemia, based on the increased TNF production and elevated TNF levels in these pathology states, as discussed above.
Antibodies to a “modulator” material which was characterized as cachectin (later found to be identical to TNF) were disclosed by Cerami et al. (EPO Patent Publication 0,212,489, Mar. 4, 1987). Such antibodies were said to be useful in diagnostic immunoassays and in therapy of shock in bacterial infections. Rubin et al. (EPO Patent Publication 0,218,868, Apr. 22, 1987) disclosed monoclonal antibodies to human TNF, the hybridomas secreting such antibodies, methods of producing such antibodies, and the use of such antibodies in immunoassay of TNF. Yone et al. (EPO Patent Publication 0,288,088, Oct. 26, 1988) disclosed anti-TNF antibodies, including mAbs, and their utility in immunoassay diagnosis of pathologies, in particular Kawasaki's pathology and bacterial infection. The body fluids of patients with Kawasaki's pathology (infantile acute febrile mucocutaneous lymph node syndrome; Kawasaki, T., Allergy 16:178 (1967); Kawasaki, T., Shonica (Pediatrics) 26:935 (1985)) were said to contain elevated TNF levels which were related to progress of the pathology (Yone et al., supra).
Other investigators have described mAbs specific for recombinant human TNF which had neutralizing activity in vitro (Liang, C-M. et al. Biochem. Biophys. Res. Comm. 137:847–854 (1986); Meager, A. et al., Hybridoma 6:305–311 (1987); Fendly et al., Hybridoma 6:359–369 (1987); Bringman, T. S. et al., Hybridoma 6:489–507 (1987); Hirai, M. et al., J. Immunol. Meth. 96:57–62 (1987); Moller, A. et al. (Cytokine 2:162–169 (1990)). Some of these mAbs were used to map epitopes of human TNF and develop enzyme immunoassays (Fendly et al., supra; Hirai et al., supra; Moller et al., supra) and to assist in the purification of recombinant TNF (Bringman et al., supra). However, these studies do not provide a basis for producing TNF neutralizing antibodies that can be used for in vivo diagnostic or therapeutic uses in humans, due to immunogenicity, lack of specificity and/or pharmaceutical suitability.
Neutralizing antisera or mAbs to TNF have been shown in mammals other than man to abrogate adverse physiological changes and prevent death after lethal challenge in experimental endotoxemia and bacteremia. This effect has been demonstrated, e.g., in rodent lethality assays and in primate pathology model systems (Mathison, J. C. et al., J. Clin. Invest. 81:1925–1937 (1988); Beutler, B. et al., Science 229:869–871 (1985); Tracey, K. J. et al., Nature 330:662–664 (1987); Shimamoto, Y. et al., Immunol. Lett. 17:311–318 (1988); Silva, A. T. et al., J. Infect. Dis. 162:421–427 (1990); Opal, S. M. et al., J. Infect. Dis. 161:1148–1152 (1990); Hinshaw, L. B. et al., Circ. Shock 30:279–292 (1990)).
To date, experience with anti-TNF mAb therapy in humans has been limited but shows beneficial therapeutic results, e.g., in arthritis and sepsis. See, e.g., Elliott, M. J. et al., Baillieres Clin. Rheumatol. 9:633–52 (1995); Feldmann M, et al., Ann. N.Y. Acad. Sci. USA 766:272–8 (1995); van der Poll, T. et al., Shock 3:1–12 (1995); Wherry et al., Crit. Care. Med. 21:S436–40 (1993); Tracey K. J., et al., Crit. Care Med. 21:S415–22 (1993).
Sequence analysis of cytokine receptors has defined several subfamilies of membrane proteins (1) the Ig superfamily, (2) the hematopoietin (cytokine receptor superfamily and (3) the tumor necrosis factor (TNF)/nerve growth factor (NGF) receptor superfamily (for review of TNF superfamily see, Gruss and Dower, Blood 85(12):3378–3404 (1995) and Aggarwal and Natarajan, Eur. Cytokine Netw., 7(2):93–124 (1996)). The TNF/NGF receptor superfamily contains at least 10 different proteins. Gruss and Dower, supra. Ligands for these receptors have been identified and belong to at least two cytokine superfamilies. Gruss and Dower, supra.
The Musculoskeletal System
The Human Musculoskeletal System is comprised of skeleton (e.g., bone), muscle, tendon, ligament, and other components of joints, which constitute the basic structural framework of the body. Together, the components of this system provide the strength, stability, frame, and elasticity necessary for movement. Additionally, the musculoskeletal system protects the internal organs, stores minerals, and produces blood.
The primary component of the musculoskeletal system is the skeleton itself. The skeleton is a highly organized connection of bones responsible for many functions, including supporting the body against gravity, providing sites for muscle attachment, producing blood cells, protecting the organs and other soft body tissues, and permitting flexible movement.
Anatomically, two types of bones can be distinguished in the skeleton: flat bones (e.g., skull bones, scapula, mandible, and ileum) and long bones (e.g., tibia, femur, and humerus). The long bone is composed of two wider extremities (e.g., the epiphyses), a cylindrical tube in the middle (e.g., the midshaft or diaphysis), and a developmental zone (e.g., the metaphysis) between them. In a growing long bone, the epiphysis and the metaphysis are separated by a layer of cartilage (e.g., epiphyseal cartilage or growth plate), responsible for the longitudinal growth of the bones. The external part of the bones is formed by a layer of calcified tissue (e.g., the cortex or compact bone). In the diaphyisis, the cortex encloses the medullary cavity, the location of the hematopoietic bone marrow. Toward the metaphysis and epiphysis, the cortex becomes progressively thinner, containing a network of thin, calcified trabeculae (e.g., trabecular bone or spongy bone) and hematopoietic bone marrow. At the center of most bones is yellow marrow, which is used to store fats. Therefore, the cortical bone fulfills mainly a mechanical and protective function, and the trabecular bone fulfills a metabolic function.
Joints are formed when two bones come together and allow for bending and movement. Tough bands of connective tissue, called ligaments, surround the joints, join the two bones together, and keep the bones properly aligned. The joint capsule is lined by a synovial membrane, which produces synovial fluid for lubricating the joint. Joints may also contain fluid-filled sacs (e.g., bursa) that reduce friction in areas where skin, muscles, tendons, and ligaments rub over bones. Most joints are freely moving synovial joints; however, some joints (e.g, vertebrae) are partly movable and allow some degree of flexibility with cartilage, or menisci, between the bones, while other joints (e.g., skull sutures) do not allow movement at all.
Composed of striated bundles of myosin and actin fibers, skeletal muscles have very long fiber-like cells that contract quickly, but only when stimulated by nerve cells. Muscle is attached to the bone by tough connective tissue, called tendons, and arranged in opposing, balancing groups around joints that facilitate balanced movement.
Although the musculoskeletal system was designed for strength and endurance, the components of this system can become worn, injured, or inflamed. These disorders can range from mild to severe and from acute to chronic. Generally, the treatment depends on the type and severity of the disorder.
Bone Metabolism
Bone is formed by collagen fibers, comprising approximately 90% type I collagen, together with non-collagenous proteins. Orientation of these collagen fibers alternates between layers giving adult bone a characteristic lamellar structure. Crystals of hydroxyapatite [3Ca3(PO4)2.(OH)2] are found on collagen fibers, within them and in the ground substance of the bone. Ground substance, primarily composed of glycoproteins and proteoglycans, is a highly anionic complex which is believed to play a role in hydroxyapatite binding and calcification of the bone tissue.
Bone is a balanced, dynamic system, constantly degrading and regenerating. Initial bone development relies on the osteoblast, a cell responsible for production of the bone matrix, i.e., collagen fibers and ground substance. After formation of the bone matrix, the osteoblast reaches the end of its secreting life and differentiates into a flat bone lining cell or an osteocyte. As calcification occurs, the osteocyte then differentiates into cortical bone or trabecular bone within small osteoclast lacunae throughout the calcified collagen fiber matrix. Blood vessels penetrate the newly calcified bone, bringing the blood supply that will form the hematopoietic bone marrow.
Bone is degraded by cells called osteoclasts that are responsible for resorption of bone matrix. Osteoclasts develop from pluripotent mononuclear precursor cells found in the bone marrow. As these cells commit to the osteoclast lineage they lose proliferative potential and eventually they fuse to form multinuclear immature osteoclasts. In the presence of bone, mature osteoclasts become polarized, develop a ruffled border and begin to resorb bone. A tight seal is formed between the osteoclast and the surface or the bone to be resorbed. The osteoclast secretes lysosomal and non-lysosomal enzymes, via its ruffled border, into the extracellular bone-resorbing compartment. The osteoclast acidifies this extracellular compartment by secreting protons. The reduced pH facilitates exposure of the bone matrix by dissolving crystals and also provides conditions suitable for the function of lysosomal enzymes in matrix degradation. Digestion products of this resorption process may be internalized by the osteoclast, transported through the osteoclast by transcytosis to be released at the basolateral domain of the cell or released through relapse of the seal created between the osteoclast and the bone surface. Bone resorption by osteoclasts serves to remove bone matrix from the center of the bone, forming the central cavity of the long bones during bone formation, and also to allow bone remodeling in the adult.
Diseases and Disorders of the Bone
Several types of bone disorders occur from an imbalance of the growth and breakdown cycles of bone. The most common, osteoporosis, is a metabolic bone disease characterized by a low bone mass and microarchitectural deterioration of bone tissue leading to progressive decrease in the density of bones, causing them to weaken. A distinguishing characteristic of osteoporosis is the normal mineral/collagen ration in affected tissues, in contrast to the disease osteomalacia in which a mineral deficiency relative to collagen is observed. Osteoporosis occurs in several different types and is seen more often in older women. Postmenopausal osteoporosis is generally found in women between the ages 51 and 75 and is caused by the lack of estrogen. Senile osteoporosis results not only from the imbalance between growth and breakdown but also from the calcium deficiency associated with age. Secondary osteoporosis is caused by secondary effects of another medical condition (e.g., chronic renal failure, hormonal disorders) or by drugs (e.g., barbiturates, anticonvulsants). Idiopathic juvenile osteoporosis is a rare form that occurs in children and young adults who, for no obvious reason, have weak bones. Treatment for all forms of osteoporosis is aimed at increasing bone density (e.g., estrogen intake, bisphosphonates, fluoride supplements).
Paget's Disease also results from an imbalance of the growth and breakdown of bone. The turnover rate is areas affected by Paget's Disease increases tremendously; resulting in abnormal, enlarged bone that is soft and weak. Although no specific genetic pattern has been determined, Paget's Disease tends to appear in family lineages. There is no direct treatment for Paget's Disease, rather treatment is given only alleviate pain and discomfort.
Bone disorders can also result from infection. Bone can be infected through three routes: bloodstream, direct invasion, and adjacent soft tissue infections. Osteomyelitis is a bone infection usually caused by bacteria (e.g., Staphylococcus aureus) which results in swelling of the soft bone marrow tissue, compression of the blood vessels, and possibly death of parts of bone. Pott's disease is an infection of the vertebrae by the bacteria that cause tuberculosis (e.g., Mycobacterium tuberculosis, M. bovis, or M. africanum.) For acute infections, antibiotics are generally the most effective treatment for this disease. However, if the infection is severe or chronic, surgery may also be required to remove the infected tissue and replaced with healthy bone, muscle, or skin.
Most bone carcinomas are benign. The most common type of benign bone tumor, usually occurring in people aged 10 to 20, is osteochrondroma. Osteochrondromas are growths on the surface of a bone that protrude as hard lumps. Benign chondromas, usually occurring in people aged 10 to 30, develop in the central part of the bone. Chrondroblastomas, usually occurring in people aged 10 to 20, are rare, painful tumors that grow in the ends of bones. Osteoid osteomas are very small tumors that commonly develop in the arms or legs but can occur in any bone. Giant cell tumors, usually occurring in people aged 20–40, most commonly originate in the ends of the bones and may extend into adjacent tissue. Treatment of these tumors generally involves pain management and, possibly, surgery to remove the tumor.
Although rare, malignant bone tumors may be primary or metastatic. In children, most malignant bone tumors are primary; in adults, most are metastatic. The most common type of malignant primary tumor, multiple myeloma, originates in the red bone marrow cells and most commonly occurs in older people. Osteosarcoma, usually occurring in people aged 10–20, commonly occurs in or around the knee and cause pain and swelling. These tumors tend to spread to the lungs. Chrondrosarcomas are slow-growing tumors composed of cancerous cartilage cells. Ewing's sarcoma, occurring most commonly in males aged 10 to 20, develop most often in arms and legs. These tumors can become large and can affect the entire length of a bone. Metastatic bone tumors most often originate from breast, lung, prostate, kidney and thyroid cancers.
Treatment for bone tumors depends on the type of cancer. Most treatments are complex and involve a combination of chemotherapy, radiotherapy, and surgery. Prompt treatment is especially important for malignant bone tumors.
Accordingly, there is a need to provide methods for use in the diagnosis and treatment of metabolic bone diseases and disorders. Such methods may also be used in the diagnosis, prognosis and treatment of diseases and/or disorders associated with metabolic bone diseases and disorders and/or aberrant osteoclast development and/or activity.